GC-MS-IRMS: Undisputable results by coupling of GC-IRMS with high-resolution mass spectrometry for final confirmation in sports drug testing

Importance of the Topic

Gas chromatography coupled with isotope ratio mass spectrometry (GC-IRMS) is a cornerstone in anti-doping analysis, providing definitive differentiation between endogenous and synthetic steroids based on their 13C/12C ratios. High confidence in compound identity and purity is critical to avoid false positives or negatives in sports drug testing.

Objectives and Study Overview

The study demonstrates a novel hyphenation of a high-resolution GC-MS system (Q Exactive GC Orbitrap) with a GC-IRMS setup to deliver simultaneous isotopic and structural information in a single run. An excretion study following oral administration of 30 mg testosterone to a volunteer was used to evaluate method performance up to 24 hours post-dose.

Methodology and Instrumentation

Urine samples underwent enzymatic hydrolysis, solid-phase and liquid-liquid extraction, followed by two HPLC cleanup steps producing seven steroid fractions. Selected fractions were acetylated to improve chromatographic behavior. A Trace 1310 GC equipped with a micro channel device split the column effluent (5:1 ratio) between:

• Delta V Plus or 253 Plus IRMS via GC IsoLink II and ConFlo IV for δ13C measurement
• Q Exactive GC Orbitrap for full-scan high-resolution accurate-mass spectra

Main Results and Discussion

– δ13C values of target compounds (testosterone, 5α- and 5β-diol metabolites) exhibited clear 3–10‰ depletion relative to endogenous reference compounds (pregnanediol, 11-ketoetiocholanolone, 5α-androst-16-en-3α-ol) throughout 24 hours.
– High mass resolution (120 000 FWHM) enabled unambiguous identification of co-eluting peaks and minor metabolites via accurate-mass spectral matching (<2 ppm error) against library entries.
– IRMS ratio trace (m/z 45/44) served as an additional purity check, revealing any isotopic disturbances due to co-elution.

Benefits and Practical Applications

• Single-run acquisition of isotopic and structural data enhances throughput and confidence in adverse analytical findings.
• High resolution MS confirms peak identity, addresses matrix interferences, and supports retrospective analysis of new markers.
• Extended detection window up to 24 hours post-administration supports anti-doping enforcement.

Future Trends and Applications

• Integration of non-targeted screening workflows using full-scan orbitrap data to discover emergent doping agents.
• Automation of isotope correction algorithms and online data fusion for real-time decision support.
• Expansion to hydrogen isotope ratio analysis and multi-element IRMS coupling for broader metabolite coverage.

Conclusion

Coupling GC-IRMS with a high-resolution GC-MS system offers a powerful, streamlined platform for comprehensive steroid analysis in sports drug testing. The combined approach ensures accurate isotopic ratio determination and unequivocal compound identification in one analytical run, strengthening the reliability of doping control.

Used Instrumentation

• Thermo Scientific Trace 1310 GC with micro channel splitter
• GC IsoLink II conversion unit and ConFlo IV interface
• Delta V Plus or 253 Plus IRMS
• Q Exactive GC Orbitrap MS
• TriPlus RSH autosampler
• Thermo Scientific TraceGold TG-5MS capillary column

References

1. WADA Technical Document TD2016IRMS.
2. Piper T. et al. Rapid Commun. Mass Spectrom. 2008, 22, 2161.
3. Piper T. et al. Drug Test. Anal. 2012, 4, 717.
4. Docherty G. et al. Rapid Commun. Mass Spectrom. 2001, 15, 730.